Bottom Line:
Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects.We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration.We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.

Results: In live cell microscopy we observed that Nrf2 undergoes autonomous translocational frequency-modulated oscillations between cytoplasm and nucleus. Oscillations occurred in quiescence and when cells were stimulated at physiological levels of activators, they decrease in period and amplitude and then evoke a cytoprotective transcriptional response. We propose a mechanism whereby oscillations are produced by negative feedback involving successive de-phosphorylation and phosphorylation steps. Nrf2 was inactivated in the nucleus and reactivated on return to the cytoplasm. Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects. The serine/threonine-protein phosphatase PGAM5, member of the Nrf2 interactome, was a key regulatory component.

Innovation: We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration. Regulation of ARE-linked protective gene transcription occurs rather through translocational oscillations of Nrf2. We discovered cytoplasmic refresh rate of Nrf2 is important in maintaining and regulating the transcriptional response and links stress challenge to increased cytoplasmic surveillance. We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.

Mentions:
To examine Nrf2 stress response signaling between cytoplasmic and nuclear compartments we studied real-time changes in subcellular localization of Nrf2 in human HMEC-1 (human microvascular endothelial cell line-1) cells in vitro by live cell time-lapse fluorescence microscopy. HMEC-1 cells were transfected to express green fluorescent protein (GFP)-Nrf2 fusion protein. Dynamics of nucleus/cell GFP-Nrf2 fluorescence intensity ratio obtained from time-lapse image sequences revealed that Nrf2 undergoes oscillatory cytoplasm-nucleus translocation. Observation periods were for 400 min (Fig. 2A and Supplementary Video S1; Supplementary Data are available online at www.liebertpub.com/ars). Total cell fluorescence was unchanged over this period—in keeping with unchanged total cellular Nrf2 protein content (see Fig. 4B, L, and N). The fluorescence was quantified by computing the mean fluorescence per pixel in the nucleus and whole cell, ĪNucleus and ĪCell respectively. Average pixel intensities are used because they are less dependent upon cell size. The ratio ĪNucleus/ĪCell showed the periodicity of the translocational oscillations (Fig. 2B). The accumulation of Nrf2 into the nucleus was slow and, after a time delay, its expulsion from the nucleus was relatively rapid. Oscillations for individual cells were asynchronous with those of other cells. The period of oscillation, median (lower–upper quartile), was: 129 (81–175) min (n=44) with amplitude 0.65 (0.35–0.87) arbitrary units (Fig. 2D).

Mentions:
To examine Nrf2 stress response signaling between cytoplasmic and nuclear compartments we studied real-time changes in subcellular localization of Nrf2 in human HMEC-1 (human microvascular endothelial cell line-1) cells in vitro by live cell time-lapse fluorescence microscopy. HMEC-1 cells were transfected to express green fluorescent protein (GFP)-Nrf2 fusion protein. Dynamics of nucleus/cell GFP-Nrf2 fluorescence intensity ratio obtained from time-lapse image sequences revealed that Nrf2 undergoes oscillatory cytoplasm-nucleus translocation. Observation periods were for 400 min (Fig. 2A and Supplementary Video S1; Supplementary Data are available online at www.liebertpub.com/ars). Total cell fluorescence was unchanged over this period—in keeping with unchanged total cellular Nrf2 protein content (see Fig. 4B, L, and N). The fluorescence was quantified by computing the mean fluorescence per pixel in the nucleus and whole cell, ĪNucleus and ĪCell respectively. Average pixel intensities are used because they are less dependent upon cell size. The ratio ĪNucleus/ĪCell showed the periodicity of the translocational oscillations (Fig. 2B). The accumulation of Nrf2 into the nucleus was slow and, after a time delay, its expulsion from the nucleus was relatively rapid. Oscillations for individual cells were asynchronous with those of other cells. The period of oscillation, median (lower–upper quartile), was: 129 (81–175) min (n=44) with amplitude 0.65 (0.35–0.87) arbitrary units (Fig. 2D).

Bottom Line:
Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects.We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration.We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.

Results: In live cell microscopy we observed that Nrf2 undergoes autonomous translocational frequency-modulated oscillations between cytoplasm and nucleus. Oscillations occurred in quiescence and when cells were stimulated at physiological levels of activators, they decrease in period and amplitude and then evoke a cytoprotective transcriptional response. We propose a mechanism whereby oscillations are produced by negative feedback involving successive de-phosphorylation and phosphorylation steps. Nrf2 was inactivated in the nucleus and reactivated on return to the cytoplasm. Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects. The serine/threonine-protein phosphatase PGAM5, member of the Nrf2 interactome, was a key regulatory component.

Innovation: We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration. Regulation of ARE-linked protective gene transcription occurs rather through translocational oscillations of Nrf2. We discovered cytoplasmic refresh rate of Nrf2 is important in maintaining and regulating the transcriptional response and links stress challenge to increased cytoplasmic surveillance. We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.